Myelodysplastic neoplasms (MDS) are heterogeneous malignancies originating in hematopoietic stem cells. In this explorative study, we carried out ultra-deep total RNA sequencing on FACS-sorted CD34+ bone marrow cells from 71 patients and eight healthy age-matched controls. We investigated the expression of circular RNAs (circRNAs), a group of noncoding RNAs produced by back-splicing of nonadjacent splice sites. Key findings were further explored in an independent cohort of 118 patients with MDS and ring sideroblasts. circRNA abundance was higher in the disease groups than in controls, and different spliceosome mutations were associated with distinct circRNA expression patterns. Expression of the proliferation-related gene MKI67 was negatively correlated with circRNA abundance. High circRNA abundance was associated with a significantly increased risk of disease progression at 3 years. The majority of the 38 circRNAs that were significantly upregulated in MDS demonstrated highly correlated expression, and many were associated with risk of leukemic progression. Furthermore, we confirmed the specificity of circZEB1 expression to cases with SF3B1 mutations. We conclude that aberrant circRNA expression is found in MDS and displays associations with disease characteristics and patient outcomes.

Mosaic chromosomal alterations (mCAs) are large somatic gains, losses and copy-neutral loss of heterozygosity identified in the peripheral blood, which represent a relatively unexplored spectrum of clonal haematopoiesis. We aimed to describe mCAs in two large Danish cohorts with extensive long-term registry-based follow-up and examine associations with haematological malignancy. We analysed genotype data from 97 752 participants from the Danish Blood Donor Study and 86 301 patients from the Copenhagen Hospital Biobank using the MoChA software. Association to haematological cancers was modelled with competing-risk methods and Cox regression, or logistic regression, via nationwide register linkage. Among 184 053 individuals, 15 459 (8%) carried autosomal mCAs and 14 322 females (17%) had loss of X, with prevalence increasing with age. In samples before or after diagnosis of haematological cancer, autosomal mCAs were detectable in 21%-62% of individuals depending on cancer subtype. Overall, the risk of haematological cancer was increased for individuals carrying autosomal mCA (hazard ratio [HR] 4.1 (95% confidence interval [CI] 3.1-5.5)), but not for individuals carrying loss of X chromosome (HR 1.1 [95% CI 0.7-1.6]). The level of malignancy risk was highly variable between different chromosomal aberrations, and cancer gene-related mCAs conveyed highest risk. Autosomal mCAs, especially those overlapping cancer gene loci, identify individuals at increased risk of haematological malignancy and may assist improvement of risk stratification and early detection.

Germline DDX41 variants are found in up to 4% of patients with acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDSs), with a higher prevalence among males and a late onset. In this study, we analyzed 647 Danish patients with suspected myeloid neoplasms to assess the frequency of germline DDX41 variants and the functional impact of variants of uncertain significance (VUSs) using a cell-based assay, CRISPR-Select. We identified 16 DDX41 variants in 30 patients, 14 of whom were confirmed or predicted as germline variants. Eight germline variants were classified as likely pathogenic/pathogenic (LP/P), and three as VUSs. We generated a monoallelic DDX41 K562 cell line and used it for CRISPR-Select assessment of variant effects on proliferation and survival. Pathogenic variants impaired proliferation, while benign variants did not. Missense variants had a more subtle effect than truncating ones, suggesting hypomorphic phenotypes. One DDX41 VUS had a significant negative effect on proliferation, suggesting it to be pathogenic, while the other two had no effect, suggesting them to be benign. These findings confirm a 2.4% frequency of LP/P DDX41 germline variants in Danish patients and demonstrate the value of CRISPR-Select in distinguishing pathogenic from benign variants.

Approximately half of patients with myelodysplastic syndromes (MDS) relapse or are refractory to hypomethylating agents (HMAs) and experience poor clinical outcomes. We previously reported improved survival in a phase 1/2 clinical trial combining the HMA guadecitabine with a programmed death-ligand 1 (PD-L1) inhibitor (atezolizumab) in HMA-relapsed or HMA-refractory (HMA-R/R) MDS, yet the biological features associated with durable responses to this combined epigenetic and immunotherapy approach remain unclear. Here, we performed integrated bulk and single-cell transcriptomic profiling of bone marrow samples from patients treated on this trial to identify molecular and cellular features associated with survival. Long-term survival was associated with the presence of immunosuppressive myeloid cells and primed dendritic cells at baseline, together with therapy-induced immune reinvigoration characterized by interferon pathway activation and expansion of effector lymphocytes. In contrast, short-term survival was associated with persistent senescence-associated inflammatory programs in CD34+ bone marrow cells and elevated expression of immunosuppressive immune checkpoint molecules. These findings suggest that chronic inflammatory and senescent microenvironmental states constrain effective immune activation despite combined epigenetic and immune checkpoint therapy. Here, we identify distinct bone marrow microenvironments associated with patient survival after combined epigenetic and immune checkpoint therapy and suggest candidate biomarkers to guide patient stratification in HMA-R/R MDS.

Bi-allelic CEBPA mutations occur in 5-15% of acute myeloid leukemia (AML) patients. The precise molecular consequences of CEBPA mutations, especially in combination with frequently co-occurring mutations in TET2 , WT1 , and GATA2, remain incompletely understood. Here, we present a robust human model of CEBPA -mutant AML through gene editing of healthy bone marrow-derived hematopoietic stem cells. Loss of the CEBPA-p42 isoform expressed in healthy cells with concomitant upregulation of the leukemic CEBPA-p30 isoform resulted in a myeloproliferative phenotype. Concurrent loss-of-function mutations in TET2 or WT1 drove full leukemic transformation, while GATA2 haploinsufficiency promoted erythroid precursor accumulation without overt AML. Single-cell transcriptomics and low-input proteomics revealed enhanced myeloid output, increased interferon signaling and elevated cholesterol biosynthesis in leukemic cells. Targeting cholesterol synthesis enhanced chemosensitivity, highlighting a potential therapeutic vulnerability, particularly relevant for CEBPA -mutant patients harboring co-mutations in TET2 or WT1 , which have poor outcomes. Induction of CEBPA-p30 by CRISPR/Cas gene editing in healthy human BM HSCs drives overt AML in vivo . TET2 and WT1 loss accelerate leukemogenesis, while GATA2 haploinsufficiency redirects differentiation toward erythroid precursors potentially driving acute erythroid leukemia. CEBPA-p30 AML exhibits cholesterol biosynthesis dependency, revealing a therapeutic vulnerability to statins.

Light chain monoclonal gammopathy of undetermined significance (LC-MGUS) is an asymptomatic precursor of multiple myeloma (MM) and related disorders defined by abnormal free light chain (FLC) testing. Recently, revised FLC reference intervals were proposed by the iStopMM study group. We evaluated their performance in a large population-based cohort. MGUS cases were identified from the Danish Lymphoid Cancer Research (DALY-CARE) data resource, including only individuals with Freelite FLC-measurements. Cases were classified by original and revised LC-MGUS criteria, hereby identifying cases only fulfilling original but not revised criteria (reclassified as normal). Outcomes were progression to MM or related disorders. Cumulative incidence of progression with death as competing risk was examined using Aalen-Johansen estimation. Annual progression risk was assessed using a person-years approach. In total, 360 individuals classified by original criteria, 215 by revised, while 150 (40%) were reclassified as normal. In the revised group, there were 21 (9.8%) progressions; 11 (5.1%) to MM and seven (3.3%) to AL amyloidosis. Only two individuals (1.3%) progressed in the reclassified group, none to MM or AL amyloidosis. For revised LC-MGUS, the 2- and 5-year cumulative incidence of progression was 5.8% and 8.9%, respectively, with an annual progression rate of 3%. This study validates the performance of the iStopMM revised LC-MGUS criteria in a clinical cohort. Applying the revised criteria reduced LC-MGUS diagnoses by 40% without missing cases progressing to MM or AL amyloidosis. These findings confirm the accuracy of the revised criteria and support their implementation to improve diagnostics and ensure follow-up of individuals truly at risk.

Second primary malignancies (SPMs) increasingly affect mantle cell lymphoma (MCL) survivors. In nationwide Danish and Swedish cohorts (2000-2020), we evaluated overall survival among MCL patients with SPMs versus matched non-lymphoma individuals with comparable malignancies and MCL patients without SPMs, respectively. Of 3094 MCL survivors, 19% in Denmark and 15% in Sweden developed an SPM, with a median of three years from MCL diagnosis to first SPM. MCL patients with SPMs had about double the mortality risk compared with non-lymphoma counterparts (pooled HR 2.1, 95% confidence interval (CI) 1.3-3.5), and worse survival than MCL patients without SPMs (pooled HR 1.6, 95% CI 1.4-1.9). In Swedish MCL patients with SPMs, deaths were attributed to subsequent hematologic malignancies (9%), solid cancers (14%), primary MCL (23%), non-cancer causes (11%), with 40% still alive. Development of SPMs in MCL is associated with substantially higher mortality, supporting long-term surveillance and proactive management of late complications.

Purpose:Myeloproliferative neoplasms (MPN) are rare hematological malignancies with long survival, making real-world evidence essential to complement clinical trial data. The Danish National Chronic Myeloid Neoplasia Registry (DMR) captures nearly all MPN patients nationwide, but its validity has not been systematically evaluated. We aimed to validate key clinical variables in the DMR through a nationwide audit. Methods:We sampled DMR entries from 372 persons registered with essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic myelofibrosis (preMF), or unclassifiable MPN (MPN-U) between 2017 and 2021 across all regions of Denmark. We validated eight main variables, including 27 subordinate variables of clinical importance from the DMR, against electronic health records using standardized audit procedures. Positive predictive value (PPV), negative predictive value (NPV), sensitivity, specificity, and accuracy were calculated. Results:Overall, the DMR showed mostly high PPV and intermediate/high NPV values. PPV was high for core diagnostic, treatment and comorbidity variables: ET (0.91), PV (0.95) and PMF (0.97), date of diagnosis (0.97), splenomegaly (0.98), JAK2V617F (0.97), CALR (0.92), hypertension (0.93), hyperlipidemia (0.90), diabetes (0.97), and common treatments (phlebotomy, hydroxyurea, interferon, ruxolitinib ≥.93). Intermediate PPV was observed for diagnoses of preMF (0.78) and MPN-U (0.77), and for the remaining comorbidity variables (0.75-0.85). Most misclassifications arose from false negatives, reflecting the automatic imputation of negative (no) for missing values for selected variables in the DMR which resulted in lower sensitivity and high PPV/specificity. Conclusion:The DMR demonstrates high validity for several clinically relevant variables and constitutes a reliable source of real-world data in MPN research and therefore, crosslinking registry variables with other data sources may enable robust epidemiological studies and unprecedented precision in real-world evidence for MPN patients. Nevertheless, variable documentation and data quality must be critically assessed to account for changes in data entry practices across time periods.

The host genetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have previously been studied based on cases from the earlier waves of the pandemic in 2020 and 2021, identifying 51 genomic loci associated with infection and/or severity. SARS-CoV-2 has shown rapid sequence evolution, increasing transmissibility, particularly for Omicron variants, which raises the question of whether this affected the host genetic factors. We performed a genome-wide association study of SARS-CoV-2 infection with Omicron variants, including more than 150,000 cases from four cohorts. We identified 13 genome-wide significant loci, of which only five were previously described as associated with SARS-CoV-2 infection. The strongest signal was a single nucleotide polymorphism in an intron of ST6GAL1, a gene affecting immune development and function, connected to three other associated loci (harboring MUC1, MUC5AC and MUC16) through O-glycan biosynthesis. Our study provides robust evidence for individual genetic variation related to glycosylation, translating into susceptibility to SARS-CoV-2 infections with Omicron variants. Genome-wide analyses identify 13 loci associated with susceptibility to infection with SARS-CoV-2 Omicron variants, including variation in ST6GAL1, previously associated with influenza susceptibility, and other genes involved in glycosylation processes.

ABSTRACT Large genomically annotated acute myeloid leukaemia (AML) datasets exist, but population-based contemporary cohorts remain scarce. Here we report clinicopathological, genomic, and outcome data from Danish AML patients. 2,512 AML patients were identified between 2015-2022, of whom 33.8% had available NGS data (NGS+). In patients ≤70 years, baseline characteristics and outcomes were comparable between NGS+ and NGS− groups. In patients >70 years, more NGS+ patients received intensive treatment, but survival was similar among intensively treated patients. The distribution of mutations varied significantly by age and sex, with older age and male sex exhibiting higher frequencies of adverse-risk gene mutations. In intensively treated NGS+ patients, ELN2017 stratified 5-year OS: 58.4% (favorable), 43.4% (intermediate), and 28.2% (adverse), with hazard ratios (HRs) of 0.63 (favorable) and 1.45 (adverse) relative to intermediate. ELN2022 yielded corresponding OS rates of 56.9%, 51.8%, and 29.7%, with HRs of 0.78 and 1.86. The two models had comparable predictive performance for OS in a time-dependent model. In conclusion, outcomes of intensively treated AML patients were comparable irrespective of NGS status, underscoring the representativeness of the REFORM-AML database for the Danish AML population. Age and male sex correlated with adverse-risk mutations, and both ELN2017 and ELN2022 robustly predicted survival.

Antibody-drug conjugates (ADCs) have emerged as promising targeted therapies in acute myeloid leukemia (AML). However, most ADCs exhibit off-target binding to normal hematopoietic stem and myeloid progenitor cells, resulting in adverse hemato-toxicity and narrow therapeutic windows, limiting their clinical application to young and fit AML patients eligible for intensive curative therapies. Proteoglycans with high levels of the glycosaminoglycan oncofetal chondroitin sulfate (ofCS), are abundantly expressed in solid cancers while being absent or lowly expressed in normal adult tissues. Here, we report high ofCS levels on bone marrow (BM) cells of AML patients and AML patient-derived xenografts (PDXs), while BM cells of healthy subjects showed low or undetectable ofCS levels. Consistently, an anti-ofCS antibody demonstrated binding and internalization into AML cells, and anti-ofCS ADCs effectively killed AML cells in vitro. Moreover, anti-ofCS ADC treatment significantly prolonged survival of AML PDXs compared to controls and was associated with low toxicity. Hence, anti-ofCS ADC could represent an effective therapy with acceptable toxicity applicable for all AML patients, including those ineligible or unresponsive to current intensive curative therapies. In conclusion, our study for the first time demonstrates that a glycosaminoglycanlike ofCS represents a druggable target for development of effective antibody-based AML therapies.

Importance:Early treatment may benefit patients with high-risk smoldering multiple myeloma (SMM), a precursor of active multiple myeloma, but inconsistent high-risk definitions may capture different disease subsets. Objective:To compare 2 high-risk SMM risk definitions: the AQUILA trial inclusion criteria and the 2/20/20 risk stratification model. Design, Setting, and Participants:This cohort study included data from 2 separate SMM cohorts and a retrospective analysis assessing progression risk. The Iceland Screens, Treats, and Prevents Multiple Myeloma (iStopMM) is a large population-based screening study in Iceland, conducted from 2016 to 2021, while the Danish Lymphoid Cancer Resource (DALY-CARE) is a nationwide clinical data collection from Denmark from 2002 to 2025. From the iStopMM cohort, individuals with SMM were detected by screening. In DALY-CARE, individuals with SMM were identified in a clinical setting. Data were analyzed from March 2025 to January 2026. Exposure:High-risk SMM by the AQUILA criteria, defined by presence of immunoparesis, monoclonal protein (M-protein) greater than 3.0 g/dL, immunoglobin A isotype, bone marrow plasma cell infiltration greater than 50%, or free light chain ratio 8 or greater. High risk by the 2/20/20 model was defined by presence of more than 1 of: M-protein greater than 2.0 g/dL, bone marrow plasma cell infiltration greater than 20%, or free light chain ratio greater than 20. Main Outcomes and Measures:Proportion of high-risk SMM in the 2 cohorts by both models and risk of progression as defined by initiation of treatment. Results:A total of 193 individuals (median [IQR] age, 70 [63-72] years; 116 [60%] female) from the iStopMM cohort and 1147 individuals (median [IQR] age, 72 [64-79] years; 598 [52%] female) from the DALY-CARE cohort were included. In the screened cohort (iStopMM), 65 participants (34%) had high-risk SMM according to the AQUILA criteria, and 15 participants (8%) had high-risk SMM according to the 2/20/20 model. In the clinical cohort (DALY-CARE), 607 participants (55%) met high-risk criteria according to AQUILA, and 219 participants (19%) had high-risk SMM according to the 2/20/20 model. For individuals with AQUILA-defined high-risk disease in the clinical cohort, the 2-year progression risk was 27.0% (95% CI, 23.3%-30.7%), with an annual progression rate of 14.5%. For individuals with high risk defined by 2/20/20, the 2-year progression risk was 44.1% (95% CI, 37.1%-51.1%), with an annual progression rate of 27.3%. Conclusions and Relevance:This cohort study found that the AQUILA trial criteria classified approximately 3 times more patients with SMM as having high risk compared with the 2/20/20 model and captured a group with substantially lower progression risk. These results suggest that the 2/20/20 model more accurately identifies a truly high-risk group with SMM, for whom early treatment is more likely to be beneficial.

Mixed phenotype acute leukaemia (MPAL) is a rare subtype of acute leukaemia possessing significant therapeutic challenges, as no standardized, evidence-based treatment regimen has been defined. In this nationwide study, we aimed to assess the effect of an acute lymphoid leukaemia (ALL)-like regimen; an acute myeloid leukaemia (AML)-like regimen; and a hybrid regimen on complete remission (CR), overall survival (OS) and event-free survival (EFS). Patients were identified through the Danish National Pathology Registry and validated according to the 2022 WHO classification. OS was estimated using the Kaplan-Meier estimator, and differences in OS were assessed with the log-rank test. Inverse probability weighting was used to balance compared groups with respect to age and calendar year. Among the 43 intensively treated WHO 2022 MPAL patients, 25 (58.1%) received ALL regimens, 10 (23.3%) received AML regimens and 8 (18.6%) received hybrid regimens. CR rates by treatment regimen were highest for hybrid regimen (87.5% (95% confidence interval (CI): 47.3%-99.7)), followed by ALL regimen (72% (95% CI: 50.6%-87.5%)) and AML regimen (40% (95% CI: 12.2%-73.8%)). Treatment with hybrid regimens consisting of a fludarabine-based approach (fludarabine, cytarabine, G-CSF and idarubicin [FLAG-IDA], fludarabine, cytarabine, G-CSF and mitoxantrone [Mito-FLAG] or fludarabine, etoposide, G-CSF, mitoxantrone and cytarabine [FLEGMA]) was associated with improved OS and EFS compared to a classic daunorubicin-cytarabine AML regimen (p = 0.02 and p = 0.002 respectively).

Background and Objectives: Mantle cell lymphoma (MCL) is a highly heterogenous disease, but an optimal prognostic biomarker in relapsed/refractory (R/R) MCL has not yet been established. The circular RNA-based risk score, circSCORE, was recently proposed as a promising prognosticator in newly diagnosed, younger patients with MCL. This study explores the prognostic potential of circSCORE in R/R MCL in both nodal (lymph node (LN)) and non-nodal tissues (bone marrow (BM)) and peripheral blood (PB)). Materials and Methods: RNA was extracted from 65 relapse samples consisting of first-relapse LN samples (n = 20) from patients who previously underwent first-line treatment in the MCL2 and MCL3 trials, and either BM (n = 34) or PB (n = 11) samples obtained from patients with R/R MCL included in the MCL6 trial, taken at trial baseline. Kaplan-Meier estimates, and Cox regressions were used to evaluate the association between circSCORE risk groups (high versus low) and outcomes. Results: Survival analyses showed significantly inferior outcomes for patients with high-risk circSCORE compared to low-risk score for both progression-free survival (PFS) (hazard ratio (HR) 1.99, p-value 0.0407) and overall survival (OS) (HR 2.29, p-value 0.0192) in the total cohort. The same tendencies were displayed when exploring the non-nodal samples only. Furthermore, circSCORE retained prognostic impact for PFS, but not OS, when adjusted for Ki67, MIPI, and TP53 mutation status. Conclusions: The circRNA-based risk score, circSCORE, displayed prognostic potential in R/R MCL along with promising application in non-nodal tissues, indicating that circSCORE, if further validated, might serve as an easily obtainable biomarker in R/R MCL.

A. Gating strategy applied on PBMCs samples evaluated by flow cytometry. CD8+ T cell memory was defined based on the expression of CD45RA and CCR7 (CD197). Cell surface phenotype (PD-1, CD39, and CD69) and intracellular activation (Ki-67) markers were quantified on CD8+ effector T cell (TEFF) based on indicated gates. B, CD8+ naïve and memory T cells in long-and short-survivors quantified before and after the treatment. C, Representative flow cytometry plots showing CD8+ T cells activation in PBMC samples from a short survivor. PD-1+ Ki-67+ expression on CD8+ T cells compared before and after the treatment.